LpSAPK9 phosphorylates and activates LpABF2 / LpABF3 to transactivate expression of chlorophyll catabolic genes and promote leaf senescence in perennial ryegrass

Leaf senescence is an active and tightly controlled process that is particularly important for perennial plants to survive over hostile environmental conditions. The phytohormone ABA plays a vital regulatory role in leaf senescence, and the subclass III SnRK2 family genes, such as SnRK2.2/2.3/2.6, are known as key components in ABA signaling. Yet, the functional roles and molecular mechanisms of subclass I SnRK2s in ABA-mediated leaf senescence remain less well understood. In this study, we characterized one subclass I SnRK2 kinase, LpSAPK9, in perennial ryegrass (Lolium perenne). LpSAPK9 was localized in both the chloroplast and the nucleus, and its gene expression was positively correlated with the process of leaf senescence. Overexpressing LpSAPK9 accelerated developmental and drought/osmotic-induced leaf senescence. The interactive proteins of LpSAPK9, including LpABF2 and LpABF3, were identified using yeast two-hybrid (Y2H) library screening and confirmed by firefly luciferase complementation assay (LCA), bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation (Co-IP) assays. Combining Y1H, in planta transactivation assay, and CUT&Tag-qPCR, we identified that LpABF2 and LpABF3 directly bound promoters of four chlorophyll catabolic genes (LpSGR, LpNYC1, LpNOL, and LpPPH) to activate their transcription. By phosphorylating the S89 and T130 amino acid residues in LpABF2 and the S122 in LpABF3, LpSAPK9 enhances the transcription activities of LpABF2/3 to promote chlorophyll degradation and leaf senescence. Interestingly, LpSAPK9 overexpression not only accelerated these senescence processes but also reduced leaf ABA content and down-regulated ABA biosynthetic genes. Collectively, our results reveal that the LpSAPK9-LpABF2/LpABF3 module coordinately regulates the expression of chlorophyll catabolic genes to control leaf senescence in perennial ryegrass.